Eritoran

In order to administer TLR4 antagonist eritoran into TG, the rats were anesthetized, and the skull was exposed and a small hole (1 mm in diameter) was drilled above the maxillary division of the left TG 6.5 mm anterior to interaural 0 and 2.3 mm lateral to the midline18 . The guide cannula was extended into the hole 9 mm below the skull surface to reach TG and was fixed to the skull with two stainless-steel screws and dental quick cure resin, using the method described by Kinuyo18 . The guide cannula were installed one week before the AP model setting. When the dental pulp were drilled open, the rats were administered vehicle (0.5 μL/day, 0.9% NaCl solution) or TLR4 antagonist eritoran (0.1 mM, 0.5 μL/day; Eisai) into TG once a day for a total of three successive days. Ibuprofen (3.0 mg/kg) was orally administered 60 min prior to the behavior test.

CD-1 mice (18–20 g) were injected i.m. with 50 µg B. asper venom, and exudate was collected 1 h and 24 h after envenomation, as described above. After that, two groups of 5 mice each were pretreated with either Eritoran (E5564; Eisai Co, Ltd., Woodcliff Lake, NJ, USA; 200 µg/100 µL i.v.) or SS (100 µL i.v.). After 1 h, mice received an intradermal injection of 50 µL of exudates collected at either 1 h or 24 h, previously incubated with antivenom to neutralize venom toxins, as described in Section 4.3. Control groups of mice were injected with Eritoran, as described, and then received an intradermal injection of 50 µL of either normal mouse plasma or antivenom. Increase in vascular permeability was assessed, as described above.

LPS (from Escherichia coli 0111:B4; Sigma, St Louis, MO, USA) was stored in a stock solution of 1 mg/ml at −20°C, and was diluted to various concentrations with serum-free culture medium when used. TLR4 antagonist eritoran was provided by Eisai, Inc., USA. ELISA kits for MMP-2, MMP-9, and VEGF were obtained from RayBiotech (Norcross, GA, USA). Synthesis of primers were made by Sangon Biotechnology (Shanghai, China).

EBOV and MARV infections of mice were performed in the animal BSL-4 (ABSL-4) containment facilities of the Galveston National Laboratory. The animal protocols for testing of Eritoran in mice were approved by the Institutional Animal Care and Use Committee of the UTMB. Eight- to 10-week-old wild-type C57BL/6 mice, TLR4^−/− mice, strain B6.B10ScN-TLR4^lps-del/JthJ (catalog no. 007227; The Jackson Laboratory) and TNFR1/2^−/− mice, strain B6.129S-Tnfrsf1a^tm1/mxTnfrs1b^tm1/mx/J (catalog no. 003243; The Jackson Laboratory), were infected with 1,000 PFU of either mouse-adapted EBOV strain Mayinga (17 (link)) or mouse-adapted MARV strain Ci67 (37 (link)) by intraperitoneal injection. All virus stocks were back titrated at time of infection to verify viral titers. Eritoran was prepared as described by the manufacturer and diluted to a final concentration of 2.33 mg/ml (Eisai). Mice received daily administration consisting of 100 μl of placebo (vehicle) or Eritoran at an effective dose of 233 μg/day. Mice were monitored twice daily from day 0 to day 14 postchallenge, followed by once-daily monitoring from day 15 to the end of the study at day 28. The disease was scored using the following parameters: dyspnea (possible scores of 0 to 5), recumbency (0 to 5), unresponsiveness (0 to 5), and bleeding/hemorrhage (0 to 5). All mice were euthanized at day 28 post-EBOV challenge.

This reports an animal study performed to achieve the project goals of determining the survival rate of a streptozotocin (STZ)-induced diabetic mouse with periodontal pathogen P. gingivalis (Pg)-LPS and TLR4 blocker eritoran (Eisai Inc., Andover, MA, USA) administration. Further, a morphological investigation of details of the promotion of diabetic nephropathy arising with the Pg-LPS administration. The studies here used control and experimental groups with nine mice in each group (2/cage). The manuscript was prepared following the ARRIVE guidelines.